Project 3: There is no proven microbicide strategy, and recent large scale trials have failed to show protection. While there are numerous strategies in the pipeline for development, it is essential that promising strategies be explored, as none to date have succeeded and each strategy in development has some potential drawbacks and challenges. Our group has successfully demonstrated the plausibility of targeting CCR5 using modified RANTES analogues as a strategy to prevent mucosal transmission of HIV infection. While these seminal studies have helped to revise the paradigm of topical prevention strategies, there remain several potential obstacles to the application of amino terminus modified RANTES analogues as topical strategies to prevent mucosal transmission of HIV infection. Many of these potential obstacles are shared by other topical HIV prevention strategies that have been proposed. The potential challenges to these strategies include: potential agonist activity of some RANTES analogues, potency, durability of effect, and cost. Thus Project #3 of this collaborative application will attempt to resolve these limitations with the following specific aims: Sp Aim #1: to determine the signaling pathways that mediate the induction of inflammatory cytokines by PSC-RANTES (and other RANTES analogues) in vaginal/cervical tissue and to disrupt these signaling pathways as a mechanism to reduce the inflammatory responses to RANTES analogues while preserving anti-HIV activity. Sp. Aim #2: to evaluate the antiviral activities (and immunologic effects) of combination strategies that may provide additive or synergistic antiviral protection together with RANTES analogues. Sp. Aim #3: to explore novel hydrogel formulation strategies that may permit more durable intravaginal exposure to RANTES analogues and other compounds. Sp. Aim #4: to develop methods for the high scale GMP grade synthesis of recombinant RANTES analogues (6P4-RANTES, and 5P12-RANTES, and 5P14-RANTES - fully recombinant agents that are as active in vitro as PSC-RANTES in terms of HIV inhibition) that will permit affordable application of this topical prevention strategy. Successful completion of these studies by this experienced team will likely result in clarifying the pathways to development of this promising strategy for topical prevention of HIV transmission.